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Biomedical subjects

R Martinez

Publications and source records attributed to R Martinez.

At least 163 records · Page 9Linked to original sources
PubMed1993

Expression, purification, and functional characterization of the DNA-binding domain of the herpes simplex virus type 1 UL9 protein.

The origin-binding protein (UL9 protein) encoded by the herpes simplex virus type 1 (HSV-1) UL9 gene plays a critical role in the initiation of viral DNA replication. The 317-amino acid C-terminal DNA-binding domain (UL9-COOH) of the UL9 protein was expressed as a fusion protein to glutathione S-transferase in bacteria. Milligram quantities of the fusion protein were purified by differential ammonium sulfate precipitation and affinity chromatography over reduced glutathione agarose. The UL9-COOH moiety was cleaved from the fusion protein by proteolysis with thrombin. The purified UL9-COOH displayed specific and stable binding activity toward its DNA recognition sequences within the HSV-1 origin of DNA replication.

Base Sequence↗
PubMed1993

Effect of fibrin glue on postlaminectomy scar formation.

An experimental study in rats has been carried out with the aim of studying the scar formation over the spinal dura in the presence of fibrin glue. Although the main action of fibrin glue is the enhancement of wound healing by increasing the fibrous tissue, our present data suggest that this re-absorbable biomaterial diminishes the epidural scar formation, at least the first two weeks after laminectomy. This finding leads us to consider the use of fibrin glue over spinal duramater when it is likely that reoperation may become necessary in a short period of time after a laminectomy.

Animals↗
PubMed1993

Gas gangrene from subcutaneous insulin administration.

A case of gas gangrene that caused intractable shoulder pain refractory to narcotics in an immunocompromised host is presented. Gas gangrene has been associated with severe trauma involving penetrating wounds, compound fractures, extensive soft-tissue injury, intramuscular injection of epinephrine, and interruption of arterial blood supply. This case describes an elderly insulin-dependent diabetic woman who developed gas gangrene in her arm and leg at the site of her subcutaneous insulin injections. The responsible organism was Clostridium septicum. Emergency medicine physicians must consider gas gangrene Clostridium infection in immunocompromised individuals without evidence of trauma who present with localized and intractable pain.

Aged↗
PubMed1993

Usefulness of the rearrangement of the bcr/abl gene in extramedullary (lymph nodes) blast crisis diagnosed in chronic myeloid leukaemia.

The introduction of molecular biological techniques in the study of chronic myeloid leukaemia (CML) allows us to show the bcr/abl gene rearrangement produced by the translocation between the c-abl proto-oncogene located in chromosome 9 and the bcr region located in chromosome 22, which constitutes the molecular alteration of Philadelphia chromosome in CML. We present the usefulness of the bcr/abl gene rearrangement study in the diagnosis of a blast crisis initially located in lymph nodes of a patient with CML. The DNA analysis allows demonstration that the lymph node neoplastic cells originate from the clone responsible for the CML, while obtaining metaphases from a lymph node for the cytogenetic study gives rise to enormous difficulties and is practically impossible if the problem is studied retrospectively based on frozen or fixed samples.

Blast Crisis↗
PubMed1993

Identification of guanine nucleotide binding proteins from Trypanosoma cruzi.

Guanine nucleotide binding proteins (GTP-binding proteins) function as transducers of signals in different cellular processes. We have identified several GTP-binding proteins in Trypanosoma cruzi by Western blot analyses. Six polypeptide bands, p20, p25, p28, p31, p37 and p38, were specifically detected in epimastigote crude extracts, using polyclonal antibodies directed against transducin (T) or the alpha-subunit of transducin (T alpha). Four of these bands, p28, p31, p37 and p38, were found in both the soluble and the particulate epimastigote fractions. On the other hand, two of the polypeptides, p20 and p25, were observed only in the particulate fraction, and were not solubilized using 0.2% Triton X-100 and 0.2% Nonidet P-40. A rat monoclonal antibody directed against the ras oncogene, immunorecognized a band with molecular mass of 20,000 daltons, in epimastigote homogenates. In view of their identical apparent molecular weight and solubilization properties, p20, recognized by anti-T or anti-T alpha antibodies, and the 20 KDa band, recognized by anti-ras antibodies, seem to correspond to the same polypeptide. [3H] GDP and [3H] GMP-PNP binding experiments revealed the presence of guanine nucleotide binding proteins in total epimastigote crude extracts, as well as, in the soluble, detergent soluble, and particulate fractions. A primary screening of a T. cruzi cDNA library with anti-T alpha antibodies, followed by secondary and tertiary screenings with anti-ras antibodies yielded six positive clones. One of these clones (Tc-ras1) contains a 600 bp insert which we believe encodes for the ras protein from T. cruzi. On a Northern blot, this cDNA hybridizes to a unique mRNA band of 2.0 Kilobases in epimastigotes.

Animals↗
PubMed1993

Effects of sympathetic stimulation on microcirculatory dynamics in patients with essential acrocyanosis. A study using mental stress.

With the aim of examining possible alterations of the neurogenic regulation of microvascular hemodynamics, the cutaneous microcirculation in 21 patients with essential acrocyanosis was studied using a laser-Doppler method both in basal conditions and during mental stress using an arithmetic test. The analysis of results showed a significant reduction in basal flow (p < 0.02) in patients with acrocyanosis in comparison to control subjects. In 15 out the 21 patients studied it was found that there was an increase in the flowmetric curve during mental stress, whereas the test caused a diminished flow in normal subjects. The altered response to sympathetic stimulus might be the result of microcirculatory stasis which would provoke an alteration in the neurogenic regulation mechanisms of microvascular dynamics.

Adolescent↗
PubMed1993

[Cutaneous microcirculation and diabetic disease. A functional and flowmetry study in subjects with diabetes mellitus type 2].

Laser-Doppler single fingertip skin blood flow has been evaluated in 41 euglycemic type II diabetic patients under basal conditions and after dynamic testing (both ischemia and thermal stress). The same subjects have also undergone tests for the assessment of the degree of autonomic nervous system (ANS) dysfunction. The results have been compared to those obtained in 38 age-matched healthy subjects. In diabetic patients: baseline flow levels were much higher; the post-ischemic flow increase was less evident; a shorter hyperemic phase followed ischemia; a longer latency period was noticed, during thermal stress, together with a lower and slower hyperemic peak level. According to the results of ANS dynamic tests, diabetic subjects were divided into 3 groups: Group 1 (subjects with negative results); Group 2 (subjects with only one positive result); Group 3 (subjects with more than one positive test). Microcirculation disturbances were more often found in Group 3. These results show that a correlation exists between diabetic microangiopathy and ANS dysfunction. They also support the hypothesis, already pointed out by other research groups, of a similar mechanism causing diabetic neurologic and vascular complications.

Adult↗
PubMed1992

The polyanion-binding domain of cytoplasmic Lys-tRNA synthetase from Saccharomyces cerevisiae is not essential for cell viability.

Cytoplasmic Lys-tRNA synthetase (LysRS) from Saccharomyces cerevisiae is a dimeric enzyme made up of identical subunits of 68 kDa. By limited proteolysis, this enzyme can be converted to a truncated dimer without loss of activity. Whereas the native enzyme strongly interacts with polyanionic carriers, the modified form displays reduced binding properties. KRS1 is the structural gene for yeast cytoplasmic LysRS. It encodes a polypeptide with an amino-terminal extension composed of about 60-70 amino acid residues, compared to its prokaryotic counterpart. This segment, containing 13 lysine residues, is removed upon proteolytic treatment of the native enzyme. The aim of the present study was to probe in vivo the significance of this amino-terminal extension. We have constructed derivatives of the KRS1 gene, encoding enzymes lacking 58 or 69 amino-terminal residues and, by site-directed mutagenesis, we have changed four or eight lysine residues from the amino-terminal segment of LysRS into glutamic acids. Engineered proteins were expressed in vivo after replacement of the wild-type KRS1 allele. The mutant enzymes displayed reduced specific activities (2-100-fold). A series of carboxy-terminal deletions, encompassing 3, 10 or 15 amino acids, were introduced into the LysRS mutants with modified amino-terminal extensions. The removal of three residues led to a 2-7-fold increase in the specific activity of the mutant enzymes. This partial compensatory effect suggests that interactions between the two extreme regions of yeast LysRS are required for a proper conformation of the native enzyme. All KRS1 derivatives were able to sustain growth of yeast cells, although the mutant cell lines displaying a low LysRS activity grew more slowly. The expression, as single-copy genes, of mutant enzymes with a complete deletion of the amino-terminal extension or with four Lys----Glu mutations, that displayed specific activities close to that of the wild-type LysRS, had no discernable effect on cell growth. We conclude that the polycationic extensions of eukaryotic aminoacyl-tRNA synthetases are dispensable, in vivo, for aminoacylation activities. The results are discussed in relation to the triggering role in in situ compartmentalization of protein synthesis that has been ascribed to the polypeptide-chain extensions that characterize most, if not all, eukaryotic aminoacyl-tRNA synthetases.

Alleles↗
PubMed1992

Triple-helix formation is compatible with an adjacent DNA-protein complex.

The effect of oligonucleotide-directed triple-helix formation on the binding of a protein to an immediately adjacent sequence has been examined. A double-stranded oligonucleotide was designed with a target site for the binding of a pyrimidine oligonucleotide located immediately adjacent to the recognition sequence for the herpes simplex virus type 1 (HSV-1) origin of replication binding protein, which is encoded by the UL9 gene of HSV-1. Since the optimal conditions for the binding of the UL9 protein and the pyrimidine oligonucleotide to the duplex DNA are markedly different, a pyrimidine oligonucleotide was designed to optimize binding affinity and specificity for the target duplex oligonucleotide. Consideration was given to length and sequence composition in an effort to maximize triple-strand formation under conditions amenable to the formation of the UL9-DNA complex. Using gel mobility shift assays, a trimolecular complex composed of duplex DNA bound to both a third oligonucleotide strand and the UL9 protein was detected, indicating that the UL9-DNA complex is compatible with the presence of a triple helix in the immediately adjacent sequences.

Animals↗
PubMed1992

Engineering mammalian aspartyl-tRNA synthetase to probe structural features mediating its association with the multisynthetase complex.

Aspartyl-tRNA synthetase from higher eukaryotes is a component of a multienzyme complex comprising nine aminoacyl-tRNA synthetases. The cDNA encoding cytoplasmic rat liver aspartyl-tRNA synthetase was previously cloned and sequenced. This work reports the identification of structural features responsible for its association within the multisynthetase complex. Mutant and chimeric proteins have been expressed in mammalian cells and their structural behavior analyzed. A wild-type rat liver aspartyl-tRNA synthetase, expressed in Chinese hamster ovary (CHO) cells, associates within the complex from CHO cells, whereas a mutant enzyme with a deletion of 34 amino acids from its amino-terminal extremity does not. A chimeric enzyme, made of the amino-terminal moiety of rat liver aspartyl-tRNA synthetase fused to the catalytic domain of yeast lysyl-tRNA synthetase, has been expressed in Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase. The fusion protein is stable in vivo, does not associate within the multisynthetase complex and cannot restore normal growth of the mutant cells. These results establish that the 3.7-kDa amino-terminal moiety of mammalian aspartyl-tRNA synthetase mediates its association with the other components of the complex. In addition, the finding that yeast lysyl-tRNA synthetase cannot replace the aspartyl-tRNA synthetase component of the mammalian complex, indicates that interactions between neighbouring enzymes also play a prominent role in stabilization of this multienzyme structure and strengthened the view that the multisynthetase complex is a discrete entity with a well-defined structural organization.

Amino Acid Sequence↗
PubMed1992

Genetic analysis of the herpes simplex virus type 1 UL9 gene: isolation of a LacZ insertion mutant and expression in eukaryotic cells.

HSV-1 host range mutants in complementation group 1-36 (hr27 and hr156) whose mutations map in the UL9 gene, encoding the origin binding protein, are unable to form plaques or synthesize viral DNA or late viral proteins when grown in nonpermissive Vero cells (Carmichael, E. P., Kosovsky, M. J., Weller, S. K., 1988, J. Virol. 62, 91-99). These defects are complemented efficiently by growth in the permissive cell line, S22, which contains the wild type version of several HSV genes including UL9. In this report the precise nature and location of the lesions in host range mutants hr27 and hr156 were determined by DNA sequencing; both mutants were found to contain identical single-base-pair substitutions at codons 309 and 311 in the UL9 open reading frame. This region lies within the putative helicase domain of the UL9 protein. The UL9 gene was disrupted by the insertion of an insertional mutagen ICP6::lacZ in which the Escherichia coli lacZ gene is expressed under control of the viral ICP6 promoter. Hr94, a viral mutant containing this insertion, does not form plaques or synthesize viral DNA when grown in Vero cells, although both defects are complemented efficiently on permissive cell lines. These results confirm that the UL9 gene product is essential for viral growth and DNA replication. Furthermore, since no detectable UL9 protein is synthesized in hr94-infected cells, this virus provides a useful genetic background for further structure-function analysis since no potentially interfering nonfunctional UL9 protein will be expressed. We have expressed the UL9 open reading frame under the control of the strong and inducible HSV-1 ICP6 promoter and have derived Vero cell lines containing variable copy numbers of the ICP6::UL9 construct. Cells whose copy number of this construct exceeded approximately 120 are unable to support efficient plaque formation by wild-type virus. Cell lines with low copy numbers of this construct are able to complement hr27, hr156, and hr94.

Amino Acid Sequence↗
PubMed1992

Standardization of network addressing in picture archiving and communications systems utilizing the ISO OSI protocols.

The establishment of a communications network requires the definition of addressing schemes to identify systems attached to the network. When it is anticipated that such a network will interconnect to, or communicate with, other similar networks it is beneficial to define standardized addressing schemes. This paper discusses the need for the standardization of network service access point (NSAP) addresses in picture archiving and communications systems (PACS) which use the open systems interconnect (OSI) communications protocols. Possible methods for establishing the necessary hierarchy of address registration authorities are discussed, along with the corresponding address formats.

Computer Communication Networks↗
PubMed1992

Intraoperative and external radiotherapy in resected gastric cancer: updated report of a phase II trial.

From September 1984 to August 1991, 48 evaluable patients with resected gastric cancer and apparent disease confined to locoregional area were treated with intraoperative electron beam boost to the celiac axis and peripancreatic nodal areas (15 Gy) and external irradiation (40 to 46 Gy in 4 to 5 weeks) including the gastric bed and upper abdominal nodal draining regions. At the time of evaluation for IORT, the disease was primary in 38 cases, recurrent but resectable in four (anastomosis), and unresectable in four (nodal). Post operative complications were reversible. Acute tolerance to the complete treatment program was acceptable. Late complications included life-threatening events: Six episodes of gastro intestinal bleeding (three of them had an arteriographic documentation of arterioenteric fistula) and nine with severe enteritis (five required reoperation). Other long-term treatment related complications were six cases of vertebral collapse. The median follow-up time for the entire group is 22 months. Locoregional recurrence/persistence of disease has been identified in five patients (three with residual and/or recurrent postsurgical tumor). Systemic tumor progression has been detected in 15 patients (11 in intra-abdominal sites). Overall actuarial survival for patients with positive or negative serosal involvement was 33% versus 56%. It is concluded that the treatment program described is able to induce a high locoregional tumor control rate (100%) when used strictly in an adjuvant setting and might control long term, a small portion of patients not amenable for curative surgery (2 out of 8 patients with confirmed residual post-surgical disease). Gastrointestinal bleeding and enteritis are findings that indicate treatment intensity at the upper limits of tissue tolerance. Assessment of long term tolerance of pancreatic parenchyma and large blood vessels (tissues included in the IRORT field) are pending for longer follow-up and the appropriate selective studies.

Humans↗